The genomic revolution has been empowered by technologies that have determined a vast pool of genetic information. While nucleic acids encode this information, it is the proteins that act on it. Proteins are incredibly diverse in their abundance and their properties, making them highly versatile for the dynamic tasks at hand but at the same time exceptionally difficult to analyze. It is for these reasons that the proteomic revolution still lags behind the genomic revolution. Indeed, the comprehensive analysis of the dynamic properties of proteins in cells is still largely beyond current capabilities. Here, we seek to revolutionize proteomics by synergistically combining improvements in established techniques with new approaches. By creating a National Center for Dynamic Interactome Research, we will be coupling an established mass spectrometry resource, cell biology laboratories, a systems biology resource, a structural biology center, and a computational biology center. We will overcome major bottlenecks in four key areas of proteomics technology. First, we will reform the production stage for generating intact macromolecular complexes, so that we will be able to freeze a tagged macromolecular complex in place, within moments of visualizing its position in the cell, and then isolate it together with all its components and neighbors. Second, we will optimize the analysis of each complex such that its macromolecular composition, structure, and interactions will be analyzed and quantified. Third, we will gather data on the time-dependent changes in complexes as they perform dynamic cellular processes. Fourth, we will develop software to integrate our data and represent in unprecedented detail the actions of the macromolecular players in dynamic subcellular assemblies. We will seek to make these techniques rapid, robust and routine by beta testing them in several experimental systems, that (i) represent key pieces of the cellular information pathway and that (ii) present specific technological roadblocks that have been generally acknowledged by the field. We will refine our technologies by determining how to overcome these roadblocks. RELEVANCE (See instructions): As part of the larger NIH roadmap, the aim of the Center will be to create new and useful tools to elucidate the dynamics of macromolecular interactions, and to spread these tools amongst the biomedical community. The Center will empower the community to assemble the kinds of detailed, dynamic representations of the interactions in the cell that will help elucidate the principles underlying all cellular processes, thus bridging the gaps between functional genomics, proteomics, structural biology and systems biology. These tools will enable researchers to delve into the molecular details of biological processes with unprecedented facility. The resulting insights have the potential to impact all areas of medical research, from fundamental discovery to pharmaceutical development.